[Analysis of blastomogenic activity of mammal carcinogens in Drosophila using the wts(P4) allele and RNA interference-induced p53 silencing].

The test for somatic mutagenesis and recombination in Drosophila is one of the widely used approaches for determination of possible carcinogenic effects of chemical compounds. The use of heterozygotes for mutant tumor suppressor gene wts enables more direct evaluation of the blastomogenic effects of chemical compounds, by tumor formation in the adult flies. This study presents evaluation of the SMART effectiveness upon the use of Drosophila heterozygotes for the wts(P4) gene, first included into the test system. The increase of the test resolution capacity compared to the literature data for the wts(P2) allele was observed. Using wts(P4) heterozygotes, a total of 20 carcinogenic compounds, and their slightly carcinogenic and noncarcinogenic analogs were tested. Specificity of the method was about 100%, and sensitivity depended on the type of the agent tested. The latter was absolute for the direct action carcinogens, with respect to carcinogens, requiring the metabolic activation. The sensitivity was elective and was limited by the presence of the enzymes capable of activating of these compounds. To increase the test sensitivity, the RNA interference-mediated silencing of the Drosophila p53 functional activity was successfully applied. Moreover, the frequency of wts tumor induction considerably increased both in spontaneous and induced mutagenesis conditions.

[Complementary oligonucleotide invasion into (CA/TG)31-repetitive region of linear and circular DNA duplexes].

(CA/TG)n-repeats belong to microsatellite DNA and are the most widespread among dinucleotide repeats in mammalian genomes, occupying 0.25% of the genome. These repeats are known to be recombination "hot spots", however the molecular mechanisms of this effect are not known. We proposed that the high frequency of recombination in the repetitive regions may be due to duplex conformational characteristics resulting from a special geometry of base-stacking contacts, which permits the initiation of an invasion process of single-stranded DNA into the duplex homologous region. Here we show for the first time a DNA-DNA interaction of oligonucleotides d(CA)10 and d(TG)10 with linear and circular duplexes containing (CA/TG)31-repeats, upon incubation at 37 degrees C in the absence of proteins. Using radioactively labeled oligonucleotides, we showed that duplex-oligonucleotide interaction intensities depend on their molar ratio at a duplex concentration 30 nM. Decreasing the duplex concentration to 3 nM did not influence the intensity of oligonucleotide invasion. It was shown that more than 1%, but much less than 10% of the duplexes participate in the interaction with oligonucleotides, assuming that one molecule of the duplex interacts with one molecule of the oligonucleotide. Analysis of the kinetics of the process reveals invasion of d(CA)10 at the first minute of its incubation with the duplex, while d(TG)10 interacts with the duplex at an even higher rate. We discuss the role of DNA conformation plasticity of (CA/TG)n-repeats in the phenomenon observed, as well as its biological significance, in particular the role of CA-microsatellites in the initiation of homologous recombination.

[Chemical cleavage of DNA with single base mismatches for detection of mutations of unknown localization].

The most promising approach for detection of random point mutations relies upon the DNA chemical cleavage near associated mismatching base pairs. In our study, the series of heteroduplexes with all types of mismatches and extrahelical nucleotide residues surrounded by both A x T and G x C pairs were performed via hybridization of 50-mer synthetic oligonucleotides differing in only one nucleotide at the central position. The chemical cleavage of DNA duplexes immobilized on magnetic beads by means of biotin-streptavidin interaction was carried out with chemicals, which able to attack only nucleobases flipped out of the base stack: potassium permanganate and hydroxylamine reacting to T and C respectively. The chemical reactivity of different mismatches was shown to correlate clearly with the target local structure in a particular sequence context. This work makes up for a deficiency in systematic study of DNA cleavage near mismatches in dependence on their type, orientation and flanking nucleotides. The model system elaborated may be applied to estimate the sensitivity of the methodology and to control the possibility of false-positive and false-negative result appearance, when different protocols for detection of DNA mutations are used. The modification of heteroduplex mixtures by potassium permanganate and hydroxylamine allows to reveal any non-canonical base pair and suggest its type and neighboring nucleotides from the nature of chemical as well as its localization from the length of cleavage products.

[Specific oligonucleotide invasion into the end of DNA duplex].

Phenomenon of the interaction of a double-stranded DNA fragment with an oligonucleotide complementary to the end of the duplex strand was demonstrated to occur via formation of three-stranded DNA structure with an oligonucleotide invasion. It was shown that oligonucleotides complementary to the duplex ends inhibit Holliday junction formation in solutions of homologous linear DNA fragments. This effect depends on the oligonucleotide concentration, sequence and their complementarity to the duplex ends. Formation of three-stranded complexes was demonstrated using radiolabeled oligonucleotides by agarose gel-electrophoresis followed by autoradiography. Analysis of three-stranded DNA structures by chemical cleavage of non-canonical base pairs revealed that oligonucleotide invades into duplex ends via a sequential displacement mechanism and that the level of the invasion may vary considerably.

[Carcinogenic components of smokeless tobacco and tobacco-free cigarettes].

The investigation deals with an assessment of carcinogenicity and mutagenicity of samples of smokeless tobacco now on the Russian market as well as ash from alternative cigarettes made of aromatic herbs. Our data showed that the levels of polycyclic aromatic hydrocarbons, volatile and tobacco-specific N-nitrosoamines complied with the standards in the producer-countries. Smokeless tobacco extracts failed to show (Ames) any mutagenic effects such as the "read-out frame shift" or "base-pair replacement" patterns. No tobacco-specific N-nitrosoamines were identified in herbal cigarettes. However, polycyclic aromatic hydrocarbons and volatile N-nitrosoamines content appeared to be identical to that of tobacco. Herbal cigarette smoke extracts mutagenicity induced by side-effects of carcinogenic substances was of similar magnitude as well.

[Sovol as an inducer of procarcinogen activating microsomal enzymes]. / Sovol kak induktor mikrosomnykh fermentov, aktiviruiushchikh prokartsinogeny.

The commercial mixture of polychlorinated biphenyls (Sovol) studied by biochemical and immunochemical methods was found to be an inducer of a wide range of cytochrome P-450 isoenzymes. The induced enzymes activated in the Ames test a series of procarcinogens differing both in structure and in target organs: benz(a)pyrene, 3-methylcholanthrene, nitrosomorpholine, dimethylnitrosamine, aflatoxin B1, orthoaminoazotoluene, 2-acetylaminofluorene, cyclophosphamide and benzidine. According to the parameters the studied Sovol is similar to Aroclor 1254 and may be recommended to be used as an inducer of microsomal enzymes in routine tests for carcinogen screening.

[Direct-action mutagens in exhausts of vehicles with diesel engines]. / Mutageny priamogo deistviia v vybrosakh transportnykh dvigatelei, rabotaiushchikh na dizel'nom toplive.

The genotoxic activity of exhausts from one-shaft gas-turbine GTE-5 engine (30 kW) and a standard D-54A diesel (40 kW) have been studied. Thus, the extracts of soot from GTE-5 and D-54A induced reversions in Salmonella typhimurium both with and without metabolic activation: furthermore, extracts of soot from GTE-5 demonstrated a higher mutagenic activity. The direct mutagenic effect of the exhausts depended neither on the presence of BP nor on the other polycyclic aromatic hydrocarbons (PAHs). Most probably, it was connected with the presence of nitro-PAHs. The need for studying the PAH content in vehicle engines' exhausts and for taking into account their effect in the control and standardization is established.

[The mutagenic activity of coal tar]. / Mutagennaia aktivnost' kamennougol'noi smoly.

Genotoxicity of tar samples collected at the Kemerovo and Altai by-product coke plants has been studied, the contribution of benz(a)pyrene (BP) into the total mutagenic activity of extracts being estimated. Both direct and indirect mutagenicity of the samples is determined in the Ames test. A direct mutagenic effect of coal tar may be attributed neither to the BP action nor to other polycyclic aromatic hydrocarbons. The onset of the His revertant induction in Salmonella typhimurium strains Ta 100 and TA 97 treated with the coal tar activated by the S-9 mixture is observed in doses containing BP an order lower than threshold of its activity found in sole experiment. The highest mutagenic effect of the activated coal tar is observed at a dose containing the minimal active sole dose of BP (0.72-1.12 micrograms per plate).

[The role of duplex ends in the spontaneous interaction of homologous linear DNA fragments]. / Rol' kontsevykh uchastkov dupleksov v spontannom vzaimodeistvii gomologichnykh lineinykh fragmentov DNK.

The spontaneous interaction of homologous linear DNA fragments was studied with a model of purified PCR products by agarose gel electrophoresis. To interact, duplexes required not only homology of internal regions, but also complementary ends. Fragments differing in terminal sequences did not interact. The yield of Holliday junctions (HJ), the simplest product of DNA-DNA interaction, depended on dissociation of fragment ends. Compared with genomic fragments, those with low-melting AT ends interacted with each other more efficiently and those with high-melting GC ends, less efficiently. Incubation temperature affected the equilibrium HJ concentration in solution of homologous fragments. A conclusion was made that HJ formation is initiated by nucleation of dissociated duplex ends.

[Reparative DNA synthesis in human and murine embryonic liver cell cultures treated with N-methyl-N'-nitro-N-nitrosoguanidine]. / Reparativnyi sintez DNK v kul'ture kletok émbrional'noi pecheni cheloveka i myshi, obrabotannykh N-metil-N'-nitro-N-nitrosoguanidinom.

The carcinogenic agents--UV-light and N-methyl-N'-nitro-N-nitroso-guanidine (NMNG) were shown to induce DNA repair synthesis in human and mouse liver cell cultures. The DNA repair synthesis, measured autoradiographically, was less active in liver cells than in fibroblasts from the same embryo. The hepatocytes exhibited a higher resistance to the toxic effect of both UV-light and NMNG. This phenomenon is suggested to be due to the active non-excision DNA repair in liver cells.

[The effect of transposon P(GUS . p53.259H)on the frequency of tumor clones in Drosophila melanogaster wtsp2/+ heterozygotes]. / Vliianie transpozona P(GUS . p53.259H) na chastoty opukholevykh klonov u geterozigot wtsp2/+ Drosophila melanogaster.

We showed that transposon P(GUS.p53.259H), mapped to chromosome 3 and carrying a dominant mutation p53(259)H.GUS, has a positive effect on the frequency of spontaneous and carcinogen-induced tumor mosaic clones warts- in Drosopila melanogaster heterozygotes for the tumor suppressor gene warts located in the same chromosome. The transposon effect could be explained either by the arrest of apoptosis in the cells expressing mutant p53(259)H.GUS gene and containing carcinogen-induced pre-mutations, and/or by genetic instability introduced into chromosome 3 by the P(GUS.p53.259H) transposon itself. The effect of the P(GUS.p53.259H) appeared to be carcinogen-specific. It substantially increased the frequency of tumors induced by supermutagenic platinum complex, oxoplatin, and did not increase the frequencies of tumors induced by polycyclic aromatic hydrocarbons, benzo(alpha)pyrene and pyrene. In the spectrum of mutations induced by all carcinogens tested, somatic recombination events prevailed over somatic mutations. Hence, carcinogen-specificity of the P(GUS.p53.259H) effect cannot be explained by preferential induction of somatic mutations or somatic recombination by one of the carcinogens. Organ-specificity of the increased frequency of mosaic warts- clones induced by P(GUS.p53.259H) was established.

[Induction of DNA reparative synthesis and the toxic effects of carcinogens in embryonic cell cultures]. / Induktsiia reparativnogo sinteza DNK i toksicheskie éffekty kantserogenov v kul'turakh émbrional'nykh kletok.

The carcinogens used in this work were divided into two groups according to their ability to induce DNA repair synthesis in embryonic cell cultures: powerful inducers (UV-light; N-methyl-N'-nitro-N-nitrosoguanidine; 4-nitroquinoline-1-oxide) and weak inducers (aflatoxin B1; 7,12-dimethylbenz(a)anthracene). In contrast to the latter, the former were found to be strong inhibitors of DNA replicative synthesis. To induce the active DNA repair synthesis, it is necessary to use toxic concentrations of the carcinogens examined.

[Change in the metabolism and toxic effect of benz(a)pyrene by its metabolites and a series of phenols]. / Izmenenie metabolizma i toksicheskogo deistviia benz(a)pirena s pomoshch'iu ego metabolitov i riada fenolov

Effects of some phenols and polycyclic aromatic hydrocarbon derivatives on benz(a)-pyrene metabolism have been studied in the microsomal system isolated from the mouse embryonic cell cultures. The rate of benz(a)pyrene metabolism was shown to depend on the structure and concentration of the agents added. The toxic effect of benz(a)pyrene was summed up with that of either agent studied (except ionol) added simultaneously to the cell culture.

[Genomic sequences in Drosophila melanogaster, homologous to mammalian oncogenes]. / Genomnye posledovatel'nosti Drosophila melanogaster, gomologichnye onkogenam mlekopitaiushchikh.

Drosophila melanogaster genomic sequences homologous to the mammalian oncogenes ras, src, jun, fos, ets, and wnt are considered. Their structure, expression, and functions are compared.

[The effect of hydroxymethyl derivatives of 7,12-dimethylbenz(a)anthracene on its metabolism and toxic action in tissue culture]. / Vliianie oksimetil'nykh proizvodnykh 7,12-dimetilbenz(a)antratsena i ego metabolizm i toksicheskoe deistvie v kul'ture tkani

The naturally occurring hydroxymethyl derivatives of the carcinogen 7,12-dimethylbenz(a)anthracene was found to inhibit the metabolic hydroxylation and toxic effect of this carcinogen in mouse embryo fibroblast-like cell culture. The greatest reduction of both effects was obtained when 12-hydroxymethyl-7-methylbenz(a)anthracene was added to the growth medium, less effective were, resp., 7,12-dihydroxymethylbenz(a)-anthracene and 7-hydroxymethyl-12-methylbenz(a)anthracene. The data obtained are discussed in terms of the intracellular regulation of carcinogenic hydrocarbon metabolism.

[Phenols as toxic metabolites of benz(a)pyrene]. / Fenoly kak toksicheskie metabolity benz(a)pirena.

The effect of benzo(a)pyrene and two its phenolic metabolites 3-hydroxybenzo(a)-pyrene and 6-hydroxybenzo(a)pyrene--on the cultures of normal and transformed fibroblasts has been studied. In was shown that unlike the parent carcinogen its phenolic metabolites exerted only toxic (but not transforming) effect on cultured cells, and this effect has been developed at a faster rate than that produced by benzo(a)pyrene. 6-hydroxybenzo(a)pyrene was more toxic than 3-hydroxybenzo(a) pyrene. It was concluded that both metabolites produced their effects without preliminary activation by microsomal enzymes.

[Mammalian carcinogen-induced warts tumor in Drosophilidae: a test sensitive to the blastomogenic action of chemical compounds]. / Opukholi warts u drozofily, vyzvannye kantserogenami mlekopitaiushchikh: test, chuvstvitel'nyi k blastomogennomu deistviiu khimicheskikh soedinenii.

A basically new system has been developed to screen carcinogens in Drosophilidae, which is based on somatic mutagenesis and recombination. The system may induce tumors in Drosophilidae, which are recorded in adult insects. The test uses recessive mutation in the suppressor gene of growth of warts (wts) tumors. The new system is sensitive to a wide spectrum of mutagenic carcinogens that are naturally encountered. The sensitivity of the system to polycyclic aromatic hydrocarbons and aromatic amides is higher than that of classical tests. Dominant p53 gene mutation that ceases mutagen-induced apoptosis has been shown to increase the incidence of wts tumors by many times. The magnitude of the effect depends on the rate of mutant p53 expression. The increasing effect of p53 mutation extends to both somatic recombination and point mutations and deletions at the wts locus.

[Species and tissue differences of reparative DNA synthesis in embryonic cell cultures treated with carcinogens]. / Vidovye i tkanevye razlichiia reparativnogo sinteza DNK v kul'turakh émbrional'nykh kletok, obrabotannykh kantserogenami.

DNA repair synthesis (RS) was studied in embryonic cell cultures exposed to different carcinogenic factors: UV-light, N-methyl-N-nitro-N-nitrosoguanidine, 4-nitroquinoline-1-oxide, aflatoxin BI and 7,12-dimethylbenz(a)anthracene. DNA RS level was shown to be higher in human liver cells than in murine ones. Tissue-dependent differences in DNA RS of cells damaged by carcinogens were found, too. RS-activity was higher in human, mouse and rat fibroblast cultures than in liver cultures of the same species. RS level in human kidney cultures was similar to that in human fibroblasts. The said differences should be taken into account in the evaluation of the results of testing of chemical agents for carcinogenicity, using their ability to cause DNA repair synthesis.

[Effect of 7,12-dimethylbenz(alpha)anthracene metabolites on its capacity to induce skin tumors in mice]. / Vliianie metabolitov 7,12-dimetilbenz(al'fa)antratsena na ego sposobnost' indutsirovat' opukholi kozhi u myshei

Induction of skin tumors by 7,12-dimethylbenz(a)anthracene (DMBA) in the presence of its metabolites-7-hydroxymethyl-12-methylbenz(a)anthracene (7-OHM-12-MBA) and 7,12-dihydroxymethylbenz(a)anthracene (7,12-diOHMBA) has been studied in mice. The skin of mice was treated repeatedly with benzene or acetone solutions of DMBA (22 mug in two droplets) or with the same amount of DMBA solution together with one of the above mentioned metabolites (the molecular ratio 1 : 1 or 1 : 0.5). Neither of the metabolites affected the carcinogenic activity of DMBA under the given conditions. 7,8-benzoflavone, an inhibitior of the DMBA metabolism, strongly suppressed DMBA tumorigenesis under the same experimental conditions. Whereas the effect of benz(a)-anthracene, an inducer of aryl hydrocarbon hydroxylase activity, was less pronounced.

[DNA reparative synthesis in liver cell cultures from mice with various susceptibilities to spontaneous hepatic carcinogenesis]. / Reparativnyi sintez DNK v kul'turakh kletok pecheni myshei s razlicnoi predraspolozhennost'iu k spontannomu gepatokantserogenezu.

Ultra-violet radiation-induced unscheduled DNA synthesis in liver cell cultures from CBA, C57Bl/6j and AKR adult male mice was studied autoradiographically. Ultra-violet radiation (254 nm; 1.5-36 J/m2) triggered on an active unscheduled DNA synthesis in all cell cultures studied. The levels of unscheduled DNA synthesis in the liver cell nuclei of the spontaneous hepatocarcinogenesis-susceptible strain (CBA) and relatively, resistant ones (C57Bl/6j and AKR) were similar. It is suggested that the susceptibility to hepatocarcinogenesis in different murine strains is determined by such parameters as DNA damage and repair (stage of initiation) as well as factors peculiar to the stage of promotion.